The Multiplexed Protein Levels assay is a type of immunostaining procedure used in high-throughput microscopy screening to analyze multiple protein levels in a single sample using sequential immunostaining protocols. This assay is also known as Iterative Indirect Immunofluorescence Imaging (4i) or Cyclic Immunofluorescence (CycIF).
The assay involves the following steps:
- Immunostaining: Cells are labeled with specific antibodies, each tagged with a different fluorophore, allowing for precise detection of multiple proteins of interest.
- Fluorescence Microscopy: The sample is imaged using fluorescence microscopy, and the fluorescence intensity emitted by each fluorophore is measured.
- Iterative Analysis: The strength of this assay lies in its iterative nature. After each round of imaging, the previously used antibodies are either removed or antibody-conjugated fluorophores are bleached. New antibodies are then introduced to enable the analysis of additional proteins of interest.
This versatile assay is widely applied in screening large libraries of compounds or drugs to assess their impact on cellular protein levels. It is a valuable tool for identifying potential candidates in cancer therapies and various other applications, offering a comprehensive view of complex cellular signaling pathways and protein interactions.
Assay aummary
| 4i | CycIF | |
| Working principle | This is advanced microscopy technique for analysing multiple protein levels in a single sample through sequential immunostaining. After each round, old antibodies are removed (stripped) for the analysis of additional proteins | This is advanced microscopy technique for analysing multiple protein levels in a single sample through sequential immunostaining. After each round, the fluorophores conjugated to the old antibodies are chemically bleached for the analysis of additional proteins |
| What is detected | up to 3 different protein targets per each cycle | up to 3 different protein targets per each cycle |
| Signal type | fluorescence | fluorescence |
| Platform | Automated fluorescence microscope | Automated fluorescence microscope |
| Sensitivity | 4/4 | 4/4 |
| Throughput | High | High |
| End-point/real time | End-point | End-point |
| Multiplexing | Yes, maximal protein number tested in single sample >40 | Yes, maximal protein number tested in single sample 15 |
| Model system | Adherent 2D cell cultures | Adherent 2D cell cultures |
Example output
